Orosornucoid (OMD; alphal-acid glycoprotein) is a small (K -42,000) glycoprotein with five N-linked complex glycan chains that comprise -45% of its mass. OM[D is a positive acute phase reactant in humans, and during inflammation and many disease states, the glycan composition changes. The glycosylation patterns OMD are being established for the normal state and the variance in thealternate gene product and in disease states are being established. The basis for variation in the glycosylation patterns, particularly with regard to the expression of the Lewis' structure, is being determined in order to gain further information about the interaction of OMD with E-selectin and the physiological role of OMD. Questions to be answered include: (1) is there a preference for specific glycans, (2) for specific branches in glycans and (3) for specific glycosylation sites for the inflammation-induced expression of SLe, and (4) is there a difference in (1), (2) and (3) between acute inflammation andrheumatoid arthritis. Previously in this collaboration, we have established the asialoglycan compositions at each of the five glycosylation sites, and documented how these have changed in a vaiiety of diseases. We also found that the two major gene products (I and 11) of OMD were differentially glycosylated, although we were not able to determine the extent of the differences at aH sites. Applications of the methods developed for profiling the glycosylation patterns of OMD in different individuals and in relation to the nature and course of disease states should lead to clinical assays that can be used to foHow disease states and to increased understanding of the role and control of glycosylation on circulating proteins. The continuing coflaboration concentrates on two related areas: 1) We have purified gene product I and presently have H at -80% purity by sequence. The differences in glycosylation at each site wiH be determined. OMD is under-sialylated, and these analyses will be done on the sialylated forms of the proteins to provide the distribution of NeuNAc among the sites. 2) The serum concentration of ONO and the glycoform distribution at individual sites change in disease, as does the extent of sialylation. In particular, the amount of sialyl Lewis X antigen carried by OMDincreases in inflarnmation. We shall follow the course of occurrence of SLex at each glycosylation site as a function of inflammation through the acute and chronic stages, and during treatment. These data will be compared with the extent of interaction of leucocytes with the inflamed endothelium, since tl~iis is mediated via the SLex structure and E-selectin. The structural analyses will be performed on a series of subpopulations of OMD that differ in their glycoform content based on lectin fractionation. In the Amsterdam laboratory, glycofon-ns of AGP differing in fucosylation and diantennary glycan content are and will be isolated from sera of patients under various inflammatory conditions. A selection of these glycoforms is" being analyzed for their glycan composition in total and perglycosylation sites, in collaboration with the Cincinnati and Boston groups.